High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature.

Human induced pluripotent stem cells (hiPSCs) are reprogrammed from adult or progenitor somatic cells and must make substantial adaptations to ensure genomic stability in order to become "embryonic stem cell- (ESC-) like." The DNA damage response (DDR) is critical for maintenance of such genomic integrity. Herein, we determined whether cell of origin and reprogramming method influence the DDR of hiPSCs. We demonstrate that hiPSCs derived from cord blood (CB) myeloid progenitors (i.e., CB-iPSC) via an efficient high-fidelity stromal-activated (sa) method closely resembled hESCs in DNA repair gene expression signature and irradiation-induced DDR, relative to hiPSCs generated from CB or fibroblasts via standard methods. Furthermore, sa-CB-iPSCs also more closely resembled hESCs in accuracy of nonhomologous end joining (NHEJ), DNA double-strand break (DSB) repair, and C-MYC transcriptional signatures, relative to standard hiPSCs. Our data suggests that hiPSCs derived via more efficient reprogramming methods possess more hESC-like activated MYC signatures and DDR signaling. Thus, an authentic MYC molecular signature may serve as an important biomarker in characterizing the genomic integrity in hiPSCs.

Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

Cation exchange on the surface of gold nanorods with a polymerizable surfactant: polymerization, stability, and toxicity evaluation.

Gold nanorods were synthesized using a seed-mediated wet chemical approach with a quaternary ammonium surfactant, cetyltrimethylammonium bromide (CTAB), that forms a bilayer on the surface of the nanorods. The CTAB molecules in the bilayer were exchanged with a similar polymerizable analog, 11-(acryloyloxy) undecyltrimethyl ammonium bromide (p-CTAB). Mass spectrometric analysis of the degree of exchange of CTAB for p-CTAB, after gold digestion, gave 77 +/- 3 and 23 +/- 1% for p-CTAB and CTAB, respectively. On-rod polymerization with a cationic free-radical initiator was confirmed by FTIR analysis and did not induce aggregation as judged by ultraviolet-visible spectroscopy, transmission electron microscopy, and dynamic light scattering measurements after polymerization. In contrast to the nanorods before polymerization, the nanorods with a polymerized bilayer showed improved stability against dialysis as well as enhanced biocompatibility as measured using a viability assay on cultured human cells. Our results indicate that (1) CTAB molecules on the surface of the gold nanorods are exchangeable with similar surfactants that have a positively charged headgroup and (2) surfactant polymerization on the surface of the gold nanorods enhances both the stability and biocompatibility of these nanomaterials, probably by decreasing the degree of surfactant desorption from the surface.

Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects.

Gold nanorods of different aspect ratios are prepared using the growth-directing surfactant, cetyltrimethylammonium bromide (CTAB), which forms a bilayer on the gold nanorod surface. Toxicological assays of CTAB-capped nanorod solutions with human colon carcinoma cells (HT-29) reveal that the apparent cytotoxicity is caused by free CTAB in solution. Overcoating the nanorods with polymers substantially reduces cytotoxicity. The number of nanorods taken up per cell, for the different surface coatings, is quantitated by inductively coupled plasma mass spectrometry on washed cells; the number of nanorods per cell varies from 50 to 2300, depending on the surface chemistry. Serum proteins from the biological media, most likely bovine serum albumin, adsorb to gold nanorods, leading to all nanorod samples bearing the same effective charge, regardless of the initial nanorod surface charge. The results suggest that physiochemical surface properties of nanomaterials change substantially after coming into contact with biological media. Such changes should be taken into consideration when examining the biological properties or environmental impact of nanoparticles.